1. Field of the Invention
The present invention relates to fluorometers for detecting a particular fluorescence from a specimen. Specifically, the present invention relates to a time gated fluorometer for forming an output signal preferably representative of an image of the fluorescent specimen and to a specific method to enhance the analysis of the output signal received to thereby enhance the detection of the particular fluorescence.
2. Description of the Prior Art
In general, prior art fluorometers all suffer from a common problem of providing for a discrimination between the generated fluorescent signal and the background noise. Certain types of conventional fluorometers discriminate between the fluorescent signal and the background noise on the basis of wave length. This type of discrimination is generally not sufficient for many types of fluorescent signals.
Another type of discrimination can be accomplished using a time gated technique. In particular, these instruments are based on the principle of permitting the observation of the fluorescence or luminescence a short, and if desired a variable, time after the excitation period. Time gated fluorometers therefore add an additional level of discrimination by viewing the signal fluorescence during an optimal time window. In the past, this technique generally employed a fluorophore of long decay time in order to allow the background fluorescence to decay.
The time gated technique is in general based on a phosphoroscope invented by Becquerel in 1867. In the Becquerel instrument, the luminescent substance is placed between two rotating discs which are mounted on a common axis and which have sector shaped apertures. The variable time gating is achieved by an adjustment of the angle between a sector on one disc and a sector on the other. Subsequent refinements of the time gating technique have been accomplished by the use of spark discharges, oscilloscopes, Kerr cells and supersonic cells.
The rotating disc invented by Becquerel was put into a conical configuration for a microscope by Jones as described in U.S. Pat. No. 2,071,408 in 1937. Other more recent improvements have used electronic techniques. For example, Wieder, U.S. Pat. No. 4,341,957, provided for the gating of a detecting circuit electronically and used a laser for excitation. In this way, as in other refinements of Becquerel phosphoroscope, the gating mechanism may be adjusted so that observation of the desired signal can be optimized. Other prior art devices such as Mueller, U.S. Pat. No. 4,006,360, use electronic gating to distinguish between species of differing decay time where two species are involved and one is bound dye and the other is an unbound dye.
Two commercial instruments are currently available for the measurement of decay time or lifetimes. Both of these instruments utilize nanosec. flash sources (electric spark in air at reduced pressure). One instrument puts the output of a photomultiplier tube onto a fast ocilloscope. Provision is made to match the experimental curve with a sum of up to 3 or 4 exponentials.
The second instrument excites the sample by repeated flashes from the source (such as at 5 kHz) and pulses the photomultiplier at increasingly longer times after the flash. The output is fed into a recorder or computer and gives an intensity vs. time signal. In addition, this instrument is supplied with software to reconvolute the experimental curve by a well known method termed Linearized Least Squares Reconvolution.
Both wavelength based discrimination and time based discrimination suffer by having background fluorescence superimposed on the signal with only an indirect means of segregating the two. In addition, the use of dyes of long decay time effectively smears out the desired signal over a long time period, thus making this signal hard to extract. Dyes of long decay time have inherently low extinction coefficients and therefore provide inefficient excitation of the fluorescence.